Activation of the immune system is a tightly controlled process, with dysregulation resulting in serious health consequences such as autoimmunity and allergies. Antigen presenting cells (APC), including dendritic cells, represent key regulators of immune activation. However, the molecular mechanisms that govern immune priming by APC remain incompletely defined. Recently, the protein MARCH1 (membrane-associated RING-CH protein 1) was reported to influence the expression of molecules known to be essential for APC function, through ubiquitin-dependent mechanisms. This proposal will test the overall hypothesis that MARCH1 regulates the trafficking of B7.2 within antigen-presenting cells via a novel pathway to control costimulation during T cell priming. Here, we will analyze the regulation by MARCH1 of one of its substrates, the costimulatory molecule, B7.2 (CD86). B7.2 serves a critical, non- redundant role in the initial activation of T cells and in tempering ongoing T cell responses. Despite its critical function in these processes, there is relatively little known regarding the biogenesis and trafficking of B7.2. This is in contrast to MHC class II, the other known substrate of MARCH1, where it is clear that post-translational mechanisms regulate the trafficking of class II during the maturation of APC. We will utilize a comparative approach to define the pathways employed by MARCH1 to regulate B7.2 versus class II expression, as well as the mechanisms that regulate MARCH1 activity in response to maturation signals received by dendritic cells (Specific Aims 1 and 2). These experiments will employ a number of cell biological techniques to examine the biogenesis and sorting of B7.2 within dendritic cells at distinct stages of maturation. In turn, the impact of these pathways on T cell activation by dendritic cells will be determined using functional assays involving dendritic cells from MARCH1-deficient mice (Specific Aim 3). MARCH1 has emerged as an important potential regulator of antigen presentation within the immune system. As such, it is positioned to impact both immune priming and tolerance, and is thus directly relevant to human health. A detailed understanding of the mechanisms by which MARCH1 is able to regulate its substrates is lacking, though essential. This goal will be met through completion of the studies set forth in this proposal. PUBLIC HEALTH RELEVANCE: The immune system is critical for protection from infectious agents and cancer, yet it's inappropriate activation can produce very serious health consequences such as such as autoimmunity and allergies. This research proposal will study the function of a newly discovered mammalian protein (MARCH1) that appears to regulate the balance between beneficial and detrimental immune responses. Completion of these studies will provide a better understanding of the pathways that control activation of the immune system, and thereby lead to the development of more effective vaccines and new targets for improved treatment of autoimmune diseases.